Suspension container for binding particles for the isolation of biological material

ABSTRACT

A device, method and system is provided for binding particles for the separation and/or isolation of biological materials. In particular, a container is provided including a suspension of binding particles for the isolation of biological material, inner walls forming an elongate groove at the bottom of said container, and a cover having openings and/or being penetrable for a linear arrangement of multiple pipets or pipet tips, said openings being located above and parallel to said elongate groove.

FIELD OF THE INVENTION

The present invention belongs to the field of sample preparation foranalytical purposes, particularly the separation and/or isolation ofbiological materials such as nucleic acids or proteins in or fromcomplex mixtures. Within that field, the present invention relates toproviding binding particles for the separation and/or isolation ofbiological materials.

BACKGROUND OF THE INVENTION

The isolation of biological materials such as nucleic acids or proteinsfrom complex biological mixtures such as, e.g., clinical samples hasbeen of considerable significance especially for diagnostic purposes.

Numerous different methods have been developed in the art, e.g.,denaturing, precipitating and removing undesired components in a samplewith subsequent precipitation and isolation of the analyte in question(for example alcohol-based precipitation of nucleic acids).

Another approach is the binding of the biological material to beisolated to a solid support material which may be provided, e.g., in theform of chromatographic columns.

For diagnostic purposes, and especially for the automated isolation ofbiological materials subject to subsequent medium- or high-throughputanalysis, binding particles are often used. Such particles can havefunctionalized surfaces, i.e., they are often coated with antibodies,nucleic acid capture probes or the like, in order to bind the desiredanalyte. Alternatively, they may have unmodified surfaces such as glasssurfaces particularly for the isolation of nucleic acids.

Such binding particles are, in connection with automated analyzers,often provided as a suspension in a container from which they areretrieved and dispensed with the help of pipetting tools. EP 1 614 475suggests an approach for providing particles from a container for theisolation of biological material, disclosing the use of a container inthe form of a plastic bottle agitated by a shaker, and the use of apipet for distribution of the particles

The present invention uses an improved approach displaying severaladvantages.

SUMMARY OF THE INVENTION

The present invention relates to providing binding particles for theseparation and/or isolation of biological materials.

In particular, it relates to a suspension container (1), comprising asuspension of binding particles for the isolation of biologicalmaterial, inner walls (2) forming an elongate groove (3) at the bottomof said container, and a cover (4) having openings (5) and/or beingpenetrable for a linear arrangement of multiple pipets or pipet tips,said openings being located above and parallel to said elongate groove.

Further provided is a method for pipetting a suspension of bindingparticles for the isolation of biological material, said methodcomprising the automated steps of:

a. agitating a container (1) containing said suspension of bindingparticles, said container comprising inner walls (2) forming an elongategroove (3) at the bottom of said container, said container furthercomprising a cover (4) having openings (5) and/or being penetrable for alinear arrangement of multiple pipets or pipet tips;

b. introducing into said suspension said linear arrangement of multiplepipets or pipet tips through said openings and/or penetrable cover; and

c. aspirating at least a portion of said suspension with a lineararrangement of multiple pipets or pipet tips and releasing the aspiratedsuspension or a portion thereof into multiple vessels.

Also provided by the invention is an analytical system for providingbinding particles for the isolation of biological material, saidanalytical system comprising a linear arrangement of multiple pipets orpipet tips, a container containing a suspension of binding particles forbinding said biological material, said container comprising an elongatecavity at its bottom, said container further comprising a cover, saidcover having openings and/or being penetrable for said lineararrangement of multiple pipets or pipet tips, and a shaker for agitatingsaid container and suspending said binding particles.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a perspective view of a suspension container with coverfrom above.

FIG. 2 shows a perspective view of a suspension container without coverfrom above.

FIG. 3 shows a section view of a suspension container without cover.

FIG. 4 shows a perspective view of a suspension container from below.

FIG. 5 shows a perspective view of a suspension container placed on ashaking device.

FIGS. 6 a-c show a multipipettor interacting with the suspensioncontainer.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to providing binding particles for theseparation and/or isolation of biological materials.

In particular, a suspension container, a method and an analytical systemfor the purpose mentioned above are provided.

Among the advantages that certain embodiments of the present inventionprovide are, for example, the following:

-   -   homogeneous distribution of binding particles from a container        to vessels for downstream processing, particularly isolation of        biological material    -   parallelization of the provision of binding particles, thus        facilitating a high throughput especially for automated systems    -   avoiding contamination    -   increasing stability of the suspension    -   avoiding loss of suspension material due to agitation of the        container    -   quantitative retrieval of the suspension from its container    -   flexibility in throughput    -   individual dispense of binding particles.

It is set out in the following which embodiments bring about whichspecific advantages.

In a first aspect, the invention brings about certain advantagesmentioned above by providing a suspension container, comprising:

-   -   a suspension of binding particles for the isolation of        biological material    -   inner walls forming an elongate groove at the bottom of said        container    -   a cover having openings and/or being penetrable for a linear        arrangement of multiple pipets or pipet tips, said openings        being located above and parallel to said elongate groove.

The suspension container according to the invention ensures a reducedrisk of contamination due to the presence of a cover protecting thesuspension of binding particles from potential contaminating material.

In the context of isolating biological material for subsequent analysis,contamination of the sample often leads to severe consequences, inparticular with respect to clinical samples. Especially in the case ofmaterial that becomes amplified during analysis (e.g., nucleic acids),already very little amounts of contamination can lead to false positiveresults in qualitative assays, or to overestimation of a titer inquantitative assays. Both problems may have a significant impact ondiagnosis or the respective treatment of diseases.

In addition, the introduction of inhibitory substances like, e.g.,certain proteins can impede the analytical experiments such that noresults are obtained at all.

Besides, substances could be introduced that decrease the stability ofthe suspension, or have an influence on its physicochemical propertiessuch as concentration, ionic strength etc.

Furthermore, the use of a cover is considerably advantageous when thesuspension container is agitated. Agitation of the suspension of bindingparticles is often carried out in order to resuspend particles. Wheneverthe container is not moving, sedimentation of the particles takes placedue to the influence of gravity. It is, however, important to providethe suspension of binding particles in a homogeneous manner, so that inmultiple withdrawals of suspension an essentially equal number ofbinding particles is retrieved when withdrawing equal volumes ofsuspension. As a consequence, the concentration throughout thesuspension must be essentially constant during the various events ofwithdrawal.

In the prior art, containers with a suspension of binding particles forthe isolation of biological material are, e.g., agitated on a shaker ormixed with a stirrer. While a stirrer is another potential source ofcontamination, agitation introduces the problem of losing suspension dueto spilling. On the other hand, in automated solutions, placing a coveron the container is connected with the problem that withdrawal of thesuspension should be enabled in a manner as easy as possible. Forexample, removal of the cover prior to every withdrawal would require anadditional step in a method for providing a suspension of bindingparticles, and opening the container also increases the risk ofcontamination.

Thus, one of the advantages of the suspension container according to theinvention comprises a cover having openings and/or being penetrable fora linear arrangement of multiple pipets or pipet tips. This way, therisk of both spilling and contamination is considerably reduced whilethe cover need not be removed in order to withdraw the suspension ofbinding particles. The suspension container can be used for providingsuspension until it is empty without the need to open it.

Since the cover has openings and/or is penetrable for a lineararrangement of multiple pipets or pipet tips, it can be advantageouslyused for parallelization of the provision of a suspension of bindingparticles. Using multiple pipets enables the artisan to dispend thesuspension of binding particles into multiple vessels at the same time.Particularly in the context of automated solutions, a medium to highthroughput of samples is often desired. As an example, diagnostic testsof clinical samples such as blood screening in an in vitro diagnosticcontext, e.g., for blood banks, require the analysis of a high number ofsamples within a limited period of time. Also, laboratories serving ascentralized facilities for the analysis of clinical samples from severalhospitals need to quickly provide analytical results. The latter areoften important for a treatment following the analysis of the sample.Parallelization can lead to a decrease in the number of single test runsto make a diagnosis, and potentially shorter hospital stays (e.g., if adiagnosis can be provided sooner, patients requiring antimicrobialtherapy will receive it sooner and thus recover earlier). Moreover,providing a negative result sooner can avoid the overprescription ofantibiotics.

In embodiments where the cover has openings, these are located above andparallel to the elongate groove that is formed at the bottom of thecontainer by its inner walls.

In the sense of the invention, an “opening” as used in connection withthe cover is an aperture allowing a pipet tip to be introduced into thecontainer and thus withdrawing the suspension without removing thecover. In preferred embodiment of the invention, said openings areholes. Preferably, said holes are round-shaped, while they can have anyother geometrical shape allowing the passage of the linear arrangementof pipets or pipet tips.

“Penetrable” means closed, but still able to be penetrated. Morespecifically, with regard to the cover of the suspension container,“penetrable” means that the cover is in principle closed, but stillallows the passage of the linear arrangement of pipets or pipet tips. Asan example, the cover can be a septum made of rubber or a rubber-likematerial that can be pierced by a pipetting needle. A septum used in thecontext of the invention is made of essentially flexible material.Preferably, the cover being penetrable for a linear arrangement ofpipets or pipet tips is a septum. Preferably, the cover is air-proof. Anair-proof cover has the advantage of efficiently preventing introductionof contaminating material into the container.

The groove mentioned above facilitates the complete withdrawal ofsuspension from the container, as residual volumes gather in the groove.According to the invention, the groove's elongate form is adapted to thelinear arrangement of pipets or pipet tips. The latter can thus enterthe container through the openings and aspirate the residual volume fromthe groove. Thereby it is ensured that only little residual volumeremains in the container and is discarded without having been used.Especially in an automated system including a cover through which pipetsor their corresponding tips have to be introduced in the container,usually in a straight vertical line, this would be more difficultwithout the presence of such an elongate groove. For example, a residualvolume of suspension could remain on parts of the bottom of thecontainer that are not reached by the pipets, such that they would bediscarded without having been used. Furthermore, when the container isagitated in order to achieve or maintain a homogeneous distribution ofthe binding particles, “dead corners” may result in a design without anelongate groove as used in the present invention. This means that, e.g.,in a container with a flat bottom and corners at the borders of bottomand side walls, that are, e.g., orthogonal to each other, bindingparticles may be kept in said corners and are thus not reached by thestream caused by agitation of the container. Such a phenomenon may leadto an inhomogeneous distribution of the binding particles and thusvarying concentrations of the particles withdrawn by pipetting. Thelarger the container, the more likely and the more severe the problemsset out above become.

The elongate groove can, e.g., be caused by a parabolic or otherwiseconcave arrangement of the inner walls of the container. Said groove ispreferably essentially in the center of the bottom. This way, no edgesor corners are created within the container.

The elongate groove can also be formed as a result of two inner wallsbordering each other at a certain angle. This embodiment can beadvantageous for production of the container, and the relativelysharp-edged groove can serve as a well-defined cavity for the collectionof residual suspension volumes in which the pipets or pipet tips can beintroduced for efficient withdrawal of said residual suspension volumes.Furthermore, the relatively sharp-edged groove created by the two innerwalls inclined in an angle to each other introduces a baffle-likestructure which can be advantageous for providing a homogeneousdistribution of the binding particles within the suspension. Baffles areknown in the art to contribute to a homogeneous distribution insuspensions in agitated containers such as Erlenmeyer flasks.

Thus, a preferred aspect of the invention is the suspension containerdescribed above, wherein said inner walls are inclined in an angle toeach other and thereby form said elongate groove.

The “angle” (denoted “a” in FIG. 3) is larger than 0° and smaller than180°. If said walls together formed an entirely flat bottom, the anglewould be 180°, resulting in the absence of a groove. If the angle was0°, no bottom at all could be formed. Preferably, the angle is between90° and 180°.

In the context of the invention, when pipets form the linear arrangementof pipets or pipet tips, the pipets are preferably pipetting needles.Such needles are often used in connection with automated diagnosticsystems, for example because of their advantageously small diameter andthe precision with which they can be handled, e.g., by a robotic arm. Ifthe linear arrangement of pipets or pipet tips is formed by pipet tips,said pipet tips also need to be fixed to pipets in order to serve theirpurpose of pipetting the suspension comprised by the container. In thisembodiment, said pipet tips are preferably disposable pipet tips intowhich said suspension is aspirated and from which it is dispensed again.Such a pipet tip can be used several times before being discarded andreplaced. Often, but not necessarily, such pipet tips are made ofplastic. Disposable pipet tips are generally known in the art.

A “linear arrangement” means that the pipets or pipet tips are arrangedin a row, preferably in a straight line. The arrangement comprises abearing to which the corresponding pipets are fixed. Preferably, thebearing is also adapted to manipulate and move the linear arrangement ofpipets or pipet tips in all dimensions, i.e., on an x, y and z-axis.Preferably, said bearing is a robotic arm, preferably adapted tofacilitate aspirating the suspension from the container and dispensingit into receptacles.

As mentioned above, the invention provides the opportunity to achieve ahigh analytical sample throughput and quick results by parallelization.It is thus preferable that the linear arrangement of pipets or pipettips comprises at least a certain number of pipets or pipet tips,preferably at least four, more preferably at least eight, mostpreferably eight pipets or pipet tips.

“Binding particles for the isolation of biological material” are a solidphase on which biological material can be immobilized. The particles cancomprise different materials and shapes. For example, particles such asbeads or fleece with a diameter in the nano-, micro or millimeter rangecan be suitable for binding biological material. “Biological material”,in the sense of the invention, comprises all kinds of biologicalmolecules, for example proteins or nucleic acids, but also othermolecules occurring in nature or being derivatives or syntheticanalogues or variants thereof. Furthermore, the term “biologicalmaterial” comprises viruses and eukaryotic and prokaryotic cells. Oftensuch cells or viruses are bound to said binding particles viabiomolecules, especially proteins, that are present in their respectivecell membrane or capsid. For the purpose of binding biological material,it is often advantageous to modify the surface of said binding particlesby coating them with biomolecules with a specific or non-specificaffinity to the biological material to be bound. As an example, such acoating can comprise streptavidin which specifically binds to biotin.The latter is often chemically attached to biomolecules in order to bindthem to streptavidin-comprising surfaces such as surface of respectivelymodified binding particles. For the same purpose, interactions betweenhistidine tags and nickel or between antibodies and their antigens orepitopes can be exploited. In the context of nucleic acids as abiological material to be bound, so-called capture probes can beadvantageously used. These capture probes are nucleic acids themselves,mostly oligonucleotides, often with a length of about 15 to 25nucleotides, that bind to essentially complementary nucleic acidsequences by Watson-Crick base-pairing. Methods comprising bindingparticles coated with specific binders have the advantage that aspecific biological material, such as a specific protein or nucleicacid, can be isolated while other proteins or nucleic acids are notbound. This can contribute to the isolation of such a specificbiological material for analytical purposes. The skilled artisanunderstands that there is a variety of interactions between biologicalmaterial and specific coatings of binding particles that can be usedwithin the spirit of the invention. Methods for coating such bindingparticles are described in the prior art.

Particularly interesting for nucleic acid extraction purposes is theiradsorption to a glass surface although other surfaces are possible. Manyprocedures for isolating nucleic acids from their natural environmenthave been proposed in recent years by the use of their binding behaviorto glass surfaces. If unmodified nucleic acids are the target, a directbinding of the nucleic acids to a material with a silica surface ispreferred because, among other reasons, the nucleic acids do not have tobe modified, and even native nucleic acids can be bound. These processesare described in detail by various documents, e.g., in Vogelstein B. etal., Proc. Natl. Acad. USA 76 (1979).

In the sense of the invention, the terms “isolation”, “purification” or“extraction” of biological material relate to the following: Beforebiological material like nucleic acids may be analyzed in a diagnosticassay, e.g., by amplification, they typically have to be purified,isolated or extracted from biological samples containing complexmixtures of different components. Often, for the first steps, processesare used which allow the enrichment of the material. To release thecontents of cells or viral particles, they may be treated with enzymesor with chemicals to dissolve, degrade or denature the cellular walls orviral particles. This process is commonly referred to as lysis. Theresulting solution containing such lysed material is referred to aslysate. A problem often encountered during lysis is that other enzymesdegrading the component of interest, e.g., deoxyribonucleases orribonucleases degrading nucleic acids, come into contact with thecomponent of interest during the lysis procedure. These degradingenzymes may also be present outside the cells or may have been spatiallyseparated in different cellular compartments prior to lysis. As thelysis takes place, the component of interest becomes exposed to saiddegrading enzymes. Other components released during this process may,e.g., be endotoxins belonging to the family of lipopolysaccharides whichare toxic to cells and can cause problems for products intended to beused in human or animal therapy.

There is a variety of means to tackle the above-mentioned problem. It iscommon to use chaotropic agents such as guanidinium thiocyanate oranionic, cationic, zwitterionic or non-ionic detergents when nucleicacids are intended to be set free. It is also an advantage to useproteases which rapidly degrade the previously described enzymes orunwanted proteins. However, this may produce another problem as saidsubstances or enzymes can interfere with reagents or components insubsequent steps.

Enzymes which can be advantageously used in such lysis or samplepreparation processes mentioned above are enzymes which cleave the amidelinkages in protein substrates and which are classified as proteases, or(interchangeably) peptidases. Especially advantageous for the use inlysis or sample preparation processes mentioned above is the enzymeesperase, a robust protease that retains its activity at both highalkalinity and at high temperatures (EP 1 201 753).

In the sample preparation steps following the lysis step, the componentof interest is further enriched, preferably by using the bindingparticles comprised by the suspension container according to theinvention.

For instance, a procedure for binding nucleic acids entails theselective binding of nucleic acids to glass surfaces of bindingparticles in chaotropic salt solutions and separating the nucleic acidsfrom contaminants such as agarose, proteins or cell residue. To separatethe glass particles from the contaminants, the particles may be eithercentrifuged or fluids are drawn through glass fiber filters. The use ofmagnetic particles to immobilize nucleic acids after precipitation byadding salt and ethanol is more advantageous and described, e.g., inAlderton R. P. et al., S., Anal. Biochem. 201 (1992) 166-169 and PCT GB91/00212. Magnetic, porous glass is also available on the market thatcontains magnetic particles in a porous, particular glass matrix and iscovered with a layer containing streptavidin. This product can be usedto isolate biological materials, e.g., proteins or nucleic acids, ifthey are modified in a complex preparation step so that they bindcovalently to biotin. Magnetizable particular adsorbents proved to bevery efficient and suitable for automatic sample preparation.Ferrimagnetic and ferromagnetic as well as superparamagnetic pigmentsare used for this purpose.

A preferred aspect of the invention is the suspension containerdescribed above, wherein said binding particles comprise nucleic acidbinding particles. More preferably, they comprise magnetic glassparticles. Even more preferably, they comprise magnetic glass particleswith an unmodified glass surface. Also preferably, they comprisemagnetic glass particles produced by the sol-gel method. Mostpreferably, they comprise magnetic glass particles produced by thesol-gel method and having an unmodified glass surface. In a preferredembodiment, said binding particles are nucleic acid binding particles,preferably magnetic particles and/or glass particles. In a morepreferred embodiment, said binding particles are magnetic glassparticles, preferably produced by the sol-gel method and/or having anunmodified glass surface.

The most preferred magnetic particles and methods using them are thosedescribed in WO 01/37291. In brief, these magnetic glass particles are asolid dispersion of small magnetic cores in glass. They are furthermorecomparatively small and substantially spherical. The non-magnetic finecontent of a composition of the magnetic glass particles is very lowbecause of the method of their preparation. This has the effect thatsuspensions of these magnetic glass sediment slowly and can therefore beadvantageously used for processes in molecular biology which can beautomated.

A “glass” according to the present invention is understood to be anessentially amorphous material that contains silicon. Glass can containother materials such as, for example, B₂O₃ (0-30%), Al₂O₃ (0-20%), CaO(0-20%), BaO (0-10%), K₂O (0-20%), Na₂O (0-20%), MgO (0-18%), Pb₂O₃(0-15%). Glass can also contain a smaller percentage (0-5%) of a numberof other oxides such as Mn₂O₃, TiO₂, As₂O₃, Fe₂O₃, CuO, CoO, etc.

Especially preferred are glasses that are formed using the gel solprocess described in WO 96/41811 and then dried and compressed.

Particularly useful for the nucleic acid isolation in the context of theinvention is the method according to R. Boom et al. (J Clin Microbiol.28 (1990), 495-503).

After the purification or isolation of the nucleic acids potentiallyincluding a specific target nucleic acid from their naturalsurroundings, the nucleic acids or the specific target nucleic acid maybe detected.

Optionally, prior to detection, they may be amplified by techniques suchas the Polymerase Chain Reaction (PCR, described, e.g., in U.S. Pat. No.4,683,202). PCR typically employs two or more oligonucleotide primersthat bind to a selected nucleic acid template (e.g., DNA or RNA).

Nucleic acid amplification reactions apart from PCR comprise the LigaseChain Reaction (LCR; Wu D. Y. and Wallace R. B., Genomics 4 (1989)560-69; and Barany F., Proc. Natl. Acad. Sci. USA 88 (1991)189-193);Polymerase Ligase Chain Reaction (Barany F., PCR Methods and Applic. 1(1991) 5-16); Gap-LCR (WO 90/01069); Repair Chain Reaction (EP 0439182A2), 3SR (Kwoh D. Y. et al., Proc. Natl. Acad. Sci. USA 86 (1989)1173-1177; Guatelli J. C., et al., Proc. Natl. Acad. Sci. USA 87 (1990)1874-1878; WO 92/08808), and NASBA (U.S. Pat. No. 5,130,238). Further,there are strand displacement amplification (SDA), transcriptionmediated amplification (TMA), and Qβ-amplification (for a review see,e.g., Whelen A. C. and Persing D. H., Annu. Rev. Microbiol. 50 (1996)349-373; Abramson R. D. and Myers T. W., Curr Opin Biotechnol 4 (1993)41-47).

Suitable nucleic acid detection methods are known to the expert in thefield and are described in standard textbooks as Sambrook J. et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989 and Ausubel F. et al.: CurrentProtocols in Molecular Biology 1987, J. Wiley and Sons, NY. There may bealso further purification steps before the nucleic acid detection stepis carried out as, e.g., a precipitation step. The detection methods mayinclude but are not limited to the binding or intercalating of specificdyes as ethidium bromide which intercalates into the double-stranded DNAand changes its fluorescence thereafter. The purified nucleic acid mayalso be separated by electrophoretic methods optionally after arestriction digest and visualized thereafter. There are also probe-basedassays which exploit the oligonucleotide hybridization to specificsequences and subsequent detection of the hybrid. It is also possible tosequence the nucleic acid after further steps known to the expert in thefield. A useful template-dependent nucleic acid polymerase is the ZO5DNA polymerase and mutations thereof. Other template-dependent nucleicacid polymerases comprise, e.g., Taq polymerase and Tth Polymerase.

In a preferred embodiment of the invention, the suspension containerdescribed supra further comprises one or more elements selected from thegroup of:

-   -   fill-openings for filling the suspension of binding particles        into the container    -   a removable foil sealing the cover.

While, without leaving the scope of the invention, refilling thecontainer is possible, the suspension container according to theinvention is preferably disposable, i.e., it is used until no moresuspension is present within, and then the container is discarded. Thisbears the advantage that no contamination can be introduced by refillingthe container.

For primary filling of the container with the suspension of bindingparticles for the isolation of biological material, it is preferablethat fill-openings are present in the cover. Said fill-openings can haveany geometrical shape. Preferably, the fill-openings are wider than theopenings for a linear arrangement of multiple pipets or pipet tips. In apreferred embodiment, the fill-openings are used only once for fillingthe container, and then they are sealed. Preferably, they remain sealeduntil the container is discarded. Also preferably, they are sealed in asterile manner. Different materials can be used to seal saidfill-openings, e.g., plastics, metal or others. Preferably, thefill-openings are sealed with a sterile foil.

Further in connection with the avoidance of contamination, a preferredaspect of the invention is the suspension container described above,wherein said cover has openings, said openings being covered by apenetrable septum. In this embodiment, the advantages of openings and apenetrable septum are combined. The openings taken alone provide, e.g.,the advantage of allowing pipets or pipet tips to pass through anotherwise closed cover while largely excluding sources of contamination,especially in the preferred embodiment when said openings are notsignificantly larger in diameter than necessary to let the pipets orpipet tips pass. Furthermore, they can be easily introduced into acover, the cover providing a stable barrier between the interior and theexterior of the container. The septum, on the other hand, is able toessentially seal any opening such as the aperture of the containeraccording to the invention. Since a septum as used in the invention ismade of essentially flexible material such as, e.g., rubber or othersuitable materials, openings in a septum are only created when thelinear arrangement of pipets or pipet tips penetrates said septum, whileafter such penetration the created openings essentially close again. Incombination, a cover comprising openings provides a stable barrier ontop of the container according to the invention while still allowing thelinear arrangement of pipets or pipet tips to be introduced into thecontainer, whereas an additional septum contributes to a reduced risk ofcontamination by essentially sealing said openings in the cover butbeing penetrable for said pipets or pipet tips. Preferably, said septumis fixed above or underneath said openings and seals them before andpreferably also after introduction of the linear arrangements of pipetsor pipet tips. In a further preferred embodiment, the cover comprisingopenings consists of two layers of a rigid material such as preferablyplastic, and a septum sandwiched in between said layers of a rigidmaterial, thereby sealing said openings. The septum is preferably madeof rubber or a rubber-like material, such that it essentially closes thehole after the pipets or pipet tips are removed from the openings andthus the septum. “Essentially closes” in this context means that thepenetrated septum is preferably still suited to prevent the introductionof contamination. Preferably, it is still air-proof at atmosphericpressure.

Since it is important to provide the suspension container according tothe invention in a manner that contamination is avoided as far aspossible, it is preferable that it comprises a removable foil sealingthe cover. Said foil is preferably removed prior to the first use of thecontainer in order to allow the linear arrangement of pipets or pipettips to be introduced into the container and thus the suspension.Preferably, the foil is then discarded. Alternatively, said foil canremain on or underneath the cover if it is penetrable for the lineararrangement of pipets or pipet tips.

In the context of packaging the suspension container, e.g., fortransporting and providing it to its user, it can be provided in asterile and biological material-free airproof plastic packaging, whileother packaging materials may also be suitable. Preferably, suchpackaging allows the artisan to insert the container into an automatedsystem such as an apparatus without coming into physical contact withthe suspension of binding particles within the container.

Another preferred aspect of the invention is the suspension containerdescribed above, wherein said container is adapted to hold a volume ofat least 100 ml, preferably of 100 to 500 ml, more preferably of 150 to300 ml.

Especially when aiming at automated high-throughput solutions withparallelization of sample preparation and analytical reactions, it ispreferable to use a suspension container with a comparatively largequantity of suspension of binding particles. Such a container can beused for providing a suspension of binding particles for a large numberof experiments before it has to be discarded and eventually replaced. Itis preferable not to replace a container very frequently, since, e.g.,opening and closing an apparatus mostly introduces a risk ofcontamination.

Using a container with a comparatively large volume as described supraincludes the challenge that only little residual volumes should remainunused in the container despite the large inner surface. This problem issolved by the container according to the invention by the elongategroove in conjunction with the openings and/or penetrable cover asdescribed above.

Also, the problem of spilling and thus loss of suspension when agitatingthe container is solved by the invention through the cover comprised bythe container according to the invention. This is also a problemoccurring more frequently when using containers with a higher volume.

For agitation, the suspension container is preferably placed on ashaker. Laboratory shakers are known in the art and can fulfill avariety of movements suitable for resuspending potentially sedimentedbinding particles. In order to provide a stable connection betweencontainer and shaker during agitation, a preferred aspect of theinvention is the suspension container described above, wherein saidcontainer comprises one or more fastenings for reversibly fixing thecontainer to a shaker. Said fastenings can be attached to the suspensioncontainer during or after its production.

A container according to the invention can be made from differentmaterials. If, for example, the container is made of plastic, itsproduction process preferably includes injection molding, such that saidfastenings may be introduced during the production steps. The containeris preferably made of polypropylene. In a production method useful inthe context of the present invention, the container is made in atwo-step procedure. At first, the lower part of the container, i.e., thetray later containing the suspension, is made via a one-componentinjection molding process using polypropylene and a suitable moldingtool. In another step, the cover is manufactured from two components,wherein first the basic form is made of polypropylene by injectionmolding, then the material forming a septum (e.g., rubber) is added toand combined with the cover.

With regard to filling the container with the suspension of bindingparticles, it is preferred to make the suspension by first weighing thesolid material and adding a predefined amount to a suitable containerwhich is preferably already the container according to the invention,and then adding a predefined volume of the respective liquid matrix.This way, mostly even amounts of liquid and particles and thus mostlyuniform suspensions can be provided within different containers.Furthermore, adding the particles prior to the liquid first has theadvantage that clumping is avoided, which is more likely to occur if theparticles are added to the liquid. Preparing the solution within thecontainer of the invention instead of pre-creating the suspension andthen transferring the latter as such to the container bears yet anotheradvantage: In case the suspension is added as such, it has to kepthomogeneous, e.g., by agitating, before and/or while it is transferredto the container, in order to provide mostly uniform suspensions indifferent containers. This can be avoided if the filling is performed byfirst adding the binding particles as described above.

Preferably, the shaker comprises elements such as indentations orrecesses corresponding to the fastenings present on the container inorder to permit a stable fixture. Said fixture can preferably bereopened. Since the container according to the invention is preferablydisposable, it is advantageous if it can be removed from the shakerafter being empty, so it can be replaced by a new suspension containerfilled with a suspension of binding particles.

In a further preferred aspect of the invention the footprint of thesuspension container described above is oval-shaped. This shapefacilitates achieving a homogeneous particle distribution when thecontainer is agitated. Furthermore, collecting residual volumes ofsuspension and also potentially sedimented binding particles in theelongate groove at the bottom of the container is facilitated by saidoval-shaped footprint. Other geometrical shapes serving these purposesmay be employed, such as, e.g., an essentially round-shaped footprint.The combination of a footprint as described above with said elongategroove and said cover is particularly advantageous for providing ahomogeneous distribution of binding particles.

Another preferred aspect of the invention is a method for pipetting asuspension of binding particles for the isolation of biologicalmaterial, said method comprising the automated steps of

a. agitating a container (1) containing said suspension of bindingparticles, said container comprising inner walls (2) forming an elongategroove (3) at the bottom of said container, said container furthercomprising a cover (4) having openings (5) and/or being penetrable for alinear arrangement of multiple pipets or pipet tips

b. introducing into said suspension said linear arrangement of multiplepipets or pipet tips through said openings and/or penetrable cover

c. aspirating at least a portion of said suspension with a lineararrangement of multiple pipets or pipet tips and releasing the aspiratedsuspension or a portion thereof into multiple vessels.

In the context of said method, it is preferable that the agitation isstopped before step b. This way, the linear arrangement of multiplepipets or pipet tips has a better access to the suspension, and movingparts of the container cannot damage them. Alternatively, the openingsin the cover of the container are wide enough to allow pipetting throughthe cover during agitation, whereby the linear arrangement of multiplepipets or pipet tips is not touched by the rims of the opening.

When aspirating suspension, it is preferable that enough suspension isaspirated at a time to allow for multiple pipetting into severalreaction vessels. Preferably, each pipet or pipet tip releases theaspirated suspension in multiple portions and thus pipetting events.This embodiment contributes to parallelization of the method.

A preferred aspect of the invention is the method described above,wherein in step c. said linear arrangement of multiple pipets or pipettips simultaneously releases said suspension into said multiple vessels.This means that all pipets or pipet tips belonging to said lineararrangement release the aspirated suspension or a portion thereof at thesame time. This way, e.g., multiple wells of a multi- or deepwell platecan be filled with suspension at the same time.

In another preferred embodiment, the invention concerns the methoddescribed above, wherein in step c. said linear arrangement of multiplepipets or pipet tips individually releases said suspension into saidmultiple vessels by independently manipulating one or more of saidmultiple pipets or pipet tips of said linear arrangement.

This way, e.g., only selected wells of a multi- or deepwell plate can befilled with suspension at the same time. Such pipetting in a coordinatedmanner can be controlled by a controlling unit. A control unit maycomprise software for ensuring that the different components of ananalytical system work and interact correctly and with the correcttiming, e.g., moving components such as the pipets or pipet tipsbelonging to saidlinear arrangement in a coordinated manner. The controlunit may also comprise a processor running a real-time operating system(RTOS), which is a multitasking operating system intended for real-timeapplications. In other words the system processor is capable of managingreal-time constraints, i.e., operational deadlines from event to systemresponse regardless of system load. It controls in real time thatdifferent units within the system operate and respond correctlyaccording to given instructions.

A corresponding analytical system is also provided by the invention.Thus, another preferred aspect is an analytical system for providingbinding particles for the isolation of biological material, saidanalytical system comprising:

-   -   a linear arrangement of multiple pipets or pipet tips    -   a container containing a suspension of binding particles for        binding said biological material, said container comprising an        elongate cavity at its bottom, said container further comprising        a cover, said cover having openings and/or being penetrable for        said linear arrangement of multiple pipets or pipet tips    -   a shaker for agitating said container and suspending said        binding particles.

In said analytical system, the components interact in a way as describedsupra for the container and the method according to the invention.

Another preferred aspect of the invention is the analytical systemdescribed above, wherein the system further comprises a plurality ofvessels for receiving said suspension of binding particles from saidlinear arrangement of multiple pipets or pipet tips.

It is preferable that said multiple pipets or pipet tips release theaspirated suspension into multiple vessels instead of a single vessel,in order to facilitate parallelization of the provision of suspension ofbinding particles. These multiple vessels may comprise, e.g., testreagents and/or different samples from different sources, while allvessels make use of the same suspension of binding particles for theisolation of the biological material to be analyzed.

In this context, it is preferable that said linear arrangement of pipetsor pipet tips comprises at least a certain number of pipets or pipettips, preferably at least four, more preferably at least eight, mostpreferably eight pipets or pipet tips.

Another preferred aspect of the invention is the analytical systemdescribed above, said system further comprising a separation station forthe isolation of said biological material with said binding particles.

A “separation station” is a device or a component of an analyticalsystem allowing for the isolation of the binding particles from theother material present in the fluid sample. Such a separation stationcan, e.g., comprise, but is not limited to, a centrifuge, a rack withfilter tubes, a magnet, or other suitable components. In a preferredembodiment of the invention, the separation station comprises one ormore magnets. Preferably, one or more magnets are used for theseparation of magnetic particles, preferably magnetic glass particles.If, for example, the fluid sample and the binding particles are combinedtogether in the wells of a multiwell plate, then one or more magnetscomprised by the separation station can, e.g., be contacted with thefluid sample itself by introducing the magnets into the wells, or saidone or more magnets can be brought close to the outer walls of the wellsin order to attract the magnetic particles and subsequently separatethem from the surrounding liquid.

In a preferred embodiment, the separation station comprises a multiwellplate comprising vessels with an opening at the top surface of themultiwell plate and a closed bottom. The vessels comprise an upper part,a center part and a bottom part, wherein the upper part is joined to thetop surface of the multiwell plate and preferably comprises two longerand two shorter sides. The center part preferably has a substantiallyrectangular cross-section with two longer sides, wherein said vesselsare preferably aligned in rows. A continuous space is preferably locatedbetween two adjacent rows for selectively contacting at least one magnetmounted on a fixture with the side walls in at least two Z-positions.The separation station preferably further comprises a magneticseparation station comprising at least one fixture. The fixturepreferably comprises at least one magnet generating a magnetic field. Amoving mechanism is preferably present which vertically moves said atleast one fixture comprising at least one magnet at least between firstand second positions with respect to the vessels of the multiwell plate.Preferably, said at least two Z-positions of the vessels comprise theside walls and the bottom part of said vessels. The magnetic field ofsaid at least one magnet preferably draws the magnetic particles to aninner surface of the vessel adjacent to said at least one magnet whensaid at least one magnet is in said first position. The effect of saidmagnetic field is less when said at least one magnet is in said secondposition than when said at least one magnet is in said first position.Preferably, the fixture comprising said at least one magnet comprises aframe. The separation station preferably comprises a frame to receivethe multiwell plate, and latch-clips to attach the multiwell plate.Preferably, the separation station comprises two types of magnets. Asecond preferred embodiment comprises a spring which exerts a pressureon the frame comprising the magnets such that the magnets are pressedagainst the vessels of the multiwell plate. The first magnets arepreferably constructed and arranged to interact with vessels of amultiwell plate for exerting a magnetic field on a large volume ofliquid comprising magnetic particles held in said vessels. Said secondmagnets are preferably constructed and arranged to interact with vesselsof a multiwell plate for exerting a magnetic field on a small volume ofliquid comprising magnetic particles held in said vessels. Said firstand second magnets can be preferably moved to different Z-positions.

A yet further preferred aspect of the invention is the analytical systemdescribed above, said system further comprising one or more elementsselected from the group consisting of:

-   -   a reaction module containing the components of a chemical and/or        biochemical reaction    -   a detection module for detecting signals evoked by an analyte    -   a storage module for reagents and/or disposables.

A “reaction module” is a module in which a reaction such as PolymeraseChain Reaction (PCR) or hybridization of antibodies for the analysis ofthe sample or a specific analyte therein takes place. It can, e.g.,comprise a variety of vessels like tubes or plates. The outer limits orwalls of such vessels are chemically inert such that they do notinterfere with the analytical reaction taking place within. If thematerial to be analyzed is nucleic acids, there are various methodsapplicable in this context, one very significant method being thePolymerase Chain Reaction mentioned above.

A “detection module” is a module in which the detection of a signal,preferably the signal evoked by an analyte or a control, is performed. Adetection module can, e.g., be an optical detection unit for detectingthe result or the effect of the analysis procedure. An optical detectionunit may comprise a light source, e.g., a xenon lamp, optics such asmirrors, lenses, optical filters, fiber optics for guiding and filteringthe light, one or more reference channels, or a CCD camera.

A “storage module” stores the necessary reagents to bring about achemical or biological reaction important for analysis of the sample inquestion. It can also contain further components useful for the methodof the invention, e.g., disposables such as pipet tips or vessels to beused as reaction receptacles within the reaction module.

Preferably, the analytical system according to the invention furthercomprises a control unit for controlling system components.

Such a control unit is described supra in the context of the methodaccording to the invention.

It is to be understood that preferred embodiments described for thesuspension container also apply to the method and the analytical systemaccording to the invention.

DETAILED DESCRIPTION OF THE FIGURES

Depicted in FIG. 1 is an example of a suspension container (1) accordingto the invention as seen from above at an angle. In this embodiment, thecontainer comprises a frame (7) embedding the reservoir (8) containingthe suspension of binding particles (not shown). The frame furthercomprises recesses (9) for placing the container into a device such as ashaker comprising indentations fitting into said recesses. Furthermore,the frame comprises indentations (11) for fixing the container. Thereservoir comprises a knob (12) at its outer wall which can also servefor fixation or handling of the suspension container.

Also shown are inclined walls (2) which, on the inner surface of thereservoir, form an elongate groove (3) at the bottom of the container.Said groove, depicted as a dotted line, is located in a straight linebelow and parallel to openings (5) in the cover (4) of the container,said openings comprising a penetrable septum (10) through which pipetsor pipet tips can be introduced into the suspension in the reservoir.

The fill-opening (6) serves for the primary filling of the containerwith the suspension of binding particles, but is preferably sealedafterwards and not used for retrieval of the suspension in thisembodiment.

The flattened corner (18) provides the cassette with a horizontalpolarity, such that, e.g., a gripper can recognize in which orientationthe container should be inserted in a device or, e.g., placed on ashaker.

FIG. 2 is a perspective view of a suspension container without coverfrom above. Even though the cover is part of the suspension containeraccording to the invention, this depiction shows the container without acover for the sake of clarity. The frame (7) comprises an outer rim (13)and an inner rim (14), wherein the inner rim serves as a rest forsupporting the cover (not shown).

This section view in FIG. 3 depicts the interior of the containerholding the suspension of binding particles (15). The binding particles(16) are, in this depiction, homogeneously distributed within the fluidmatrix. As portions of the suspension are withdrawn from the container,the remainder of the suspension is collected in the elongate groove (3)formed by the inclined inner walls (2), so that the remaining suspensioncan be quantitatively withdrawn from the container by a lineararrangement of pipets or pipet tips.

The perspective in FIG. 4 shows the container with the frame (7), thereservoir (8), the recesses (9), indentations (11) and knobs (12) asdescribed above. Further visible from this perspective is a cross-shapedindentation (17), wherein one of the bars is arranged underneath andparallel to the inner groove (3). The cross-shaped indentation isuseful, e.g., for mounting the container on a device such as a shaker.

FIG. 5 is a perspective view of a container placed on a shaker (19). Itcan be seen that the lateral knobs (12) serve to fix the container onsaid shaker by interaction with a gripping structure on the shaker (20).Depending on the respective movements of the shaker, it can be importantthat the container is well-fixed. The indentations (9) and (17), visiblein FIG. 1 or 4, respectively, can also be useful for fixing thecontainer on the shaker.

FIG. 6 represents the interaction of the suspension container with amultipipettor (21) comprising pipetting needles (22). In 6 a, theneedles are placed outside the container in an empty condition. In 6 b,the needles are introduced into the container through the cover openingscomprising a septum, and in 6 c, the pipettor comprising the needles islowered such far that the needles reach to the elongate groove (3) atthe bottom of the container. It is shown that in this embodiment saidgroove is adapted to the interaction with the depicted multipettor, suchthat the suspension can be withdrawn in an efficient manner.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovecan be used in various combinations. All publications, patents, patentapplications, and/or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application,and/or other document were individually indicated to be incorporated byreference for all purposes.

1-9. (canceled)
 10. A method for pipetting a suspension of binding particles for the isolation of biological material, said method comprising the automated steps of: agitating a container containing said suspension of binding particles, said container comprising inner walls forming an elongate groove at the bottom of said container, said container further comprising a cover having openings and/or being penetrable for a linear arrangement of multiple pipets or pipet tips; introducing into said suspension said linear arrangement of multiple pipets or pipet tips through said openings and/or penetrable cover; and aspirating at least a portion of said suspension with a linear arrangement of multiple pipets or pipet tips and releasing the aspirated suspension or a portion thereof into multiple vessels.
 11. The method of claim 10, wherein in said linear arrangement of multiple pipets or pipet tips simultaneously releases said suspension into said multiple vessels.
 12. The method of claim 11, wherein in said linear arrangement of multiple pipets or pipet tips individually releases said suspension into said multiple vessels by independently manipulating one or more of said multiple pipets or pipet tips of said linear arrangement. 13-16. (canceled) 